Light-activatable enzymes are of interest as chemical light amplifiers and as photoactivatable therapeutic agents. As light amplifiers, the ability of a single light activated enzyme to convert thousands of molecules per minute, see generally D. Hug, 6 Photochem. Photobiol. Rev. 87 (1981), make them attractive as switches in systems such as in vitro diagnostic assays. Therapeutic uses are possible because the skin is both highly perfused by blood and accessible to light treatment. See generally J. Parrish, 77 J. Invest. Dermatology 45 (1981).
S. Varfolomeyev et al., 15 FEBS Lett. 118 (1971), describe the formation of an acyl-enzyme between .alpha.-Chymotrypsin and the p-nitrophenyl ester of p-nitro-trans-cinnamic acid (see Formula I, in which "ENZ" is chymotrypsin). ##STR2## The bond between the enzyme and the acyl group is formed with the hydroxyl group of the serine at the catalytic center of the enzyme. See also I. Berezin et al., 8 FEBS Letters 173 (1970); K. Martinek et al., 29 Photochem. and Photobiol. 637 (1979); I. Berezin et al., 2 Enzyme Microb. Technol. 150 (1980); and N. Kazanskaya et al., 5 Enzyme Microb. Technol. 209 (1983). These approaches rely solely on steric effects to differentiate photoisomers. Generally the cis-cinnamoyl adduct is more stable than the trans complex, but in some cases this difference is as low as fivefold.
A. Turner et al., 109 J. Am. Chem. Soc. 1274 (1987), describe the formation of an acyl-enzyme between .alpha.-thrombin and the trans-isomer of O-hydroxyl-.alpha.-methylcinnammic acid (see Formula II, in which ENZ is thrombin). ##STR3## The bond between the enzyme and the acyl group is formed with the hydroxyl group of the serine at the catalytic center of the enzyme. See also A. Turner et al., 110 J. Am. Chem. Soc. 244 (1988). This compound included on the acyl group a hydroxyl group as an internal nucleophile which, on photoisomerization, leads to deacylation by attack of the internal nucleophile on the carbonyl oxygen adjacent the oxygen of the serine hydroxyl group.
Inhibition of enzyme activity in the acyl-enzyme of Formula II is temporary, and enzyme activity returns after a few hours in the dark. Furthermore, the photoactivation of these enzymes is slow and requires light intensities and wavelengths such that appreciable enzyme degradation occurs during photoactivation. Hence, there is a need for new approaches in the development of light activated acyl-enzymes. The present invention is based on our continued research in this field.